Research is proposed to develop improved analytical methods for the simultaneous determination of residues of ethopabate, amprolium and principal metabolites in chicken tissues and eggs. These methods are necessary for monitoring compliance with the FDA prescribed tolerances in the interest of human health concerns. The extensive metabolism of ethopabate necessitates a determination of its metabolites after their conversion to m- phenetidine. This metabolism may explain why ethopabate residues were not detected by current methods that were specifically developed for intact ethopabate. A simple isolation method and a solid phase extraction (SPE) method will be developed utilizing cation-exchange SPE phases to separate amprolium from ethopabate or m-phenetidine. Further purification of ethopabate or m-phenetidine will be performed on a reversed-phase SPE phase. The isolated amprolium will be assayed on a cation-ion exchange HPLC column with electrochemical detection (LCED) or reversed-phase ion pair chromatography (IPC). The ethopabate or m-phenetidine will be assayed by reversed-phase HPLC using UV detection at their lambda max or by fluorescence detection for ethopabate, LCED for m-phenetidine. If satisfactory separation cannot be obtained during clean-up, mixtures of these compounds will be assayed by IPC. The effect of the drastic conditions for conversion of ethopabate metabolites to m-phenetidine on the stability of amprolium will be studied. Recovery studies of chicken tissues and eggs spiked with amprolium and ethopabate or m-phenetidine will be conducted. Validation of the methods will be made following generally accepted method validation procedures. In the event there are more than one assay method developed for any of the compounds, the best method will be selected for monitoring residues of these compounds. Qualitative confirmatory tests will be developed for ethopabate, m- phenetidine and amprolium by at least two methods (HPLC, capillary GC and mass spectrometry). Chickens will be fed with feed containing ethopabate and amprolium to determine actual residuals of these compounds in chicken tissues and eggs. Results will indicate the minimum residual amounts that can be determined in tissues and eggs as they occur in actual feeding of the chickens. Better quantitative data can be obtained than is currently available concerning residue levels of these compounds or their principal metabolites after withdrawal from chronic systemic administration. Data will indicate whether or not the 5- day withdrawal of medicated feed prior to slaughter is sufficient.